Vectors with restriction site banks IV. pJRD184, a 3793-bp plasmid vector having 43 unique cloning sites

Heusterspreute, Michel; Thi, Vinh Ha; Emery, Steve; Tournis-Gamble, Susan; Kennedy, N.; Davison, John

doi:10.1016/0378-1119(85)90327-0

Cited by 75 publications

(23 citation statements)

References 12 publications

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“…In order to simultaneously examine the influence of betI ϩ in trans (see below), the CAT activities were measured with cells which in addition to the cat fusion plasmids also carried either plasmid pTP100 (betI ϩ ) or plasmid pTP101 (betI negative control) derived from pJRD184 (20). CAT-fusion plasmids carrying a functional betI ϩ in cis were tested only together with pTP101 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…b BetI was supplied in trans from pTP100 (betI ϩ ). pTP100 (betI ϩ ) and pTP101 (control) were derived from pJRD184 (20).…”

Section: Resultsmentioning

confidence: 99%

“…Plasmid pTP100 was made by inserting an EcoRI-BglII fragment (coordinates 2096 to 4257) from plasmid pFF221 (1) into the EcoRI-HincII sites of vector pJRD184 (20). Control plasmid pTP101 was made by insertion of a 0.1-kb HincII-EcoRI fragment from the polylinker of pUC19 (Pharmacia) into the corresponding sites of pJRD184.…”

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confidence: 99%

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The complex bet promoters of Escherichia coli: regulation by oxygen (ArcA), choline (BetI), and osmotic stress

et al. 1996

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The bet regulon allows Escherichia coli to synthesize the osmoprotectant glycine betaine from choline. It comprises a regulatory gene, betI, and three structural genes: betT (choline porter), betA (choline dehydrogenase), and betB (betaine aldehyde dehydrogenase). The bet genes are regulated by oxygen, choline, and osmotic stress. Primer extension analysis identified two partially overlapping promoters which were responsible for the divergent expression of the betT and betIBA transcripts. The transcripts were initiated 61 bp apart. Regulation of the promoters was investigated by using cat (chloramphenicol acetyltransferase) and lacZ (␤-galactosidase) operon fusions. Mutation of betI on plasmid F2 revealed that BetI is a repressor which regulates both promoters simultaneously in response to the inducer choline. Both promoters remained inducible by osmotic stress in a betI mutant background. On the basis of experiments with hns and hns rpoS mutants, we conclude that osmoregulation of the bet promoters was hns independent. The bet promoters were repressed by ArcA under anaerobic growth conditions. An 89-bp promoter fragment, as well as all larger fragments tested, which included both transcriptional start points, displayed osmotic induction and BetI-dependent choline regulation when linked with a cat reporter gene on plasmid pKK232-8. Flanking DNA, presumably on the betT side of the promoter region, appeared to be needed for ArcA-dependent regulation of both promoters.Hyperosmotically stressed cells of Escherichia coli build up the cytoplasmic osmolarity by accumulation of potassium glutamate and various osmoprotectants (12). The highest osmotolerance is achieved by the accumulation of glycine betaine (hereafter called betaine) (49). Betaine can either be taken up by the ProU and ProP systems (7,8,37) or be synthesized by the Bet system, i.e., the choline-to-betaine pathway (31). Synthesis of betaine requires an external supply of choline or the intermediate metabolite betaine aldehyde. At low external concentrations, choline is mainly taken up by the high-affinity choline porter BetT (K m ϭ 8 M), whereas at higher concentrations choline is also taken up by ProU (K m ϭ 1.5 mM) (30, 50). Oxygen-dependent choline dehydrogenase (BetA) catalyzes both steps in the oxidation of choline to betaine by the way of betaine aldehyde, whereas NAD-dependent betaine aldehyde dehydrogenase (BetB) is specific for the last step (1, 31).Biochemical data have previously revealed that expression of the Bet system is reduced under anaerobic conditions. For aerobic cells, osmotic stress gives a partial induction, but for full expression, the cells also need an external supply of choline (31). Experiments with lacZ fusions showed that the regulation occurs at the level of transcription (16).The DNA sequence of the bet region has revealed that in addition to the structural genes, the bet system encodes a regulatory protein called BetI. Albeit BetI shares some sequence homology with the TetR family of bacterial regulatory proteins (29), Bet...

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Section: Resultsmentioning

confidence: 99%

“…b BetI was supplied in trans from pTP100 (betI ϩ ). pTP100 (betI ϩ ) and pTP101 (control) were derived from pJRD184 (20).…”

Section: Resultsmentioning

confidence: 99%

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The complex bet promoters of Escherichia coli: regulation by oxygen (ArcA), choline (BetI), and osmotic stress

et al. 1996

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“…1) were inserted separately into the medium-copy-number plasmid pJRD184 (15). Only the plasmid containing the 9.5-kb fragment (pJRD9.5) complemented the vancomycin-sensitive phenotype of mutant 87.6 on LB plates containing 100 g of vancomycin per ml.…”

Section: Resultsmentioning

confidence: 99%

Amplification of a novel gene, sanA, abolishes a vancomycin-sensitive defect in Escherichia coli

Rida

Caillet

1996

J Bacteriol

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We have isolated an Escherichia coli gene which, when overexpressed, is able to complement the permeability defects of a vancomycin-susceptible mutant. This gene, designated sanA, is located at min 47 of the E. coli chromosome and codes for a 20-kDa protein with a highly hydrophobic amino-terminal segment. A strain carrying a null mutation of the sanA gene, transferred to the E. coli chromosome by homologous recombination, is perfectly viable, but after two generations at high temperature (43؇C), the barrier function of its envelope towards vancomycin is defective.An essential function of the outer membrane (39), an additional bilayer located outside the peptidoglycan in gram-negative bacteria such as Escherichia coli, is to act as a selective permeability barrier (38) towards hydrophobic (52) and hydrophilic compounds if the latter are larger than 600 Da (3). The outer membrane also prevents leakage of periplasmic components and controls direct passage of secreted proteins which lack classical amino-terminal signal sequences (e.g., hemolysin) from the cytoplasm to the external medium (sec-independent secretory pathway) (47). Also the outer membrane is probably responsible for the initiation of signals ultimately resulting in changes in gene expression (32).The inner leaflet of the outer membrane is composed, like the cytoplasmic membrane, of phospholipid molecules (42), whereas its outer leaflet is a unique structure composed of lipopolysaccharide (LPS) (50), porins (36), lipoproteins (14), and small amounts of enzymes (5, 26) and other minor proteins (65). The structures and biosynthetic pathways of these components are well-known, but many features of their insertion into the outer membrane and of their function are still obscure. The barrier function of the outer membrane in relation to its structure has been studied mainly by using membrane-damaging drugs (cation chelators, dyes, detergents, and local anesthetics, etc.) which modify its permeability (24, 57) and by the isolation and characterization of mutants with altered permeability towards various antibiotics (both hydrophobic and hydrophilic), dyes, and detergents (54).Gram-positive bacteria are sensitive to vancomycin, a glycopeptide antibiotic which inhibits late steps in the extracytoplasmic synthesis of cell wall peptidoglycan (44). Although hydrophilic (35), vancomycin cannot enter gram-negative bacteria because of its large size (1.4 kDa); thus, wild-type E. coli is naturally vancomycin resistant. We have undertaken the isolation, from a library of wild-type E. coli genes inserted in a bacteriophage vector, of one or several genetic determinants which can complement the defect of a recently isolated vancomycin-sensitive mutant of E. coli. Here we report the cloning, the mapping at min 47 of the E. coli chromosome, and the nucleotide sequence of a novel gene, named sanA, which when expressed from a medium-copy-number plasmid suppresses the vancomycin susceptibility as well as some other phenotypic characteristics of this mutant. The hydrophobic na...

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“…Plasmid pDG298 carrying the spolIIG-coding sequence under the control of the spac promoter was constructed as follows: The StyI-BamHI fragment carrying the first 166 codons of spolIIG and 48 nucleotides of upstream flanking sequence (see Karmazyn-Campelli et al 1989) was cloned after filling in the StyI site in the pJRD184 vector (Heusterspreute et al 1985 (Stragier et al 1988). pDG148 and pDG298 plasmids were amplified in E. coli and used to transform B. subtilis to kanamycin resistance (10 ~g/ml), as described previously (Mason et al 1988b).…”

Section: Stratus and Plasmidsmentioning

confidence: 99%

Identification of a new sigma-factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis.

Sun¹,

Stragier²,

Setlow³

1989

Genes Dev.

178

162

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During sporulation of Bacillus subtilis, two identical genomes segregate in two compartments, the forespore and mother cell. These genomes are expressed differentially, with some genes such as sspE turned on only in the forespore. In vitro transcription of sspE was obtained only with RNA polymerase extracted from sporulating cells. Fractionation of factors associated with this enzyme and reconstitution with core RNA polymerase from vegetative cells generated an enzyme accurately transcribing sspE in vitro and led to purification of a polypeptide with the amino-terminal sequence of the spolllG product. Inactivation of spolIIG abolished expression of sspE and five other forespore-specific genes, whereas synthesis of the spolIIG product in vegetative cells rapidly turned these genes on. Therefore, spollIG encodes a o-factor, o G, which controls the expression of multiple genes in the forespore compartment.

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Vectors with restriction site banks IV. pJRD184, a 3793-bp plasmid vector having 43 unique cloning sites

Cited by 75 publications

References 12 publications

The complex bet promoters of Escherichia coli: regulation by oxygen (ArcA), choline (BetI), and osmotic stress

The complex bet promoters of Escherichia coli: regulation by oxygen (ArcA), choline (BetI), and osmotic stress

Amplification of a novel gene, sanA, abolishes a vancomycin-sensitive defect in Escherichia coli

Identification of a new sigma-factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis.

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